1. Field of the Invention
The present invention relates generally to the treatment of cancer. More particularly, it concerns novel compounds useful for chemotherapy, methods of synthesis of these compounds and methods of treatment employing these compounds. These novel drugs comprise two main classes of compounds; one bearing modified substituents at the C-3xe2x80x2 sugar moiety and the other bearing modifications at the C-4xe2x80x2 sugar moiety. In addition, some of these analogs might also be modified at the aglycon and/or sugar moiety. These novel anthracycline analogs display high anti-tumor activity and can be used as potent drugs active against multi-drug resistant tumors. These compounds are related to other anti-tumor anthracyclines such as daunorubicin, idarubicin, epirubicin, and doxorubicin. The cytotoxic potency of these new compounds is significantly higher when compared to doxorubicin.
2. Description of Related Art
Resistance of tumor cells to the killing effects of chemotherapy is one of the central problems in the management of cancer. It is now apparent that at diagnosis many human tumors already contain cancer cells that are resistant to standard chemotherapeutic agents. Spontaneous mutation toward drug resistance is estimated to occur in one of every 106 to 107 cancer cells. This mutation rate appears to be independent of any selective pressure from drug therapy, although radiation therapy and chemotherapy may give rise to additional mutations and contribute to tumor progression within cancer cell populations (Goldie et al., 1979; Goldie et al., 1984; Nowell, 1986). The cancer cell burden at diagnosis is therefore of paramount importance because even tumors as small as 1 cm (109 cells) could contain as many as 100 to 1,000 drug-resistant cells prior to the start of therapy.
Selective killing of only the tumor cells sensitive to the drugs leads to an overgrowth of tumor cells that are resistant to the chemotherapy. Mechanisms of drug resistance include decreased drug accumulation (particularly in multi-drug resistance), accelerated metabolism of the drug and other alterations of drug metabolism, and an increase in the ability of the cell to repair drug-induced damage (Curt el al., 1984; and Kolate, 1986). The cells that overgrow the tumor population not only are resistant to the agents used but also tend to be resistant to other drugs, many of which have dissimilar mechanisms of action. This phenomenon, called pleiotropic drug resistance or multi-drug resistance (MDR), may account for much of the drug resistance that occurs in previously treated cancer patients. The development of drug resistance is one of the major obstacles in the management of cancer. One of the traditional ways to attempt to circumvent this problem of drug resistance has been combination chemotherapy.
Combination drug therapy is the basis for most chemotherapy employed to treat breast, lung, and ovarian cancers as well as Hodgkin""s disease, non-Hodgkin""s lymphomas, acute leukemias, and carcinoma of the testes. Combination chemotherapy uses the differing mechanisms of action and cytotoxic potentials of multiple drugs.
Although combination chemotherapy has been successful in many cases, the need still exists for new anti-cancer drugs. These new drugs could be such that they are useful in conjunction with standard combination chemotherapy, or these new drugs could attack drug resistant tumors by having the ability to kill cells of multiple resistance phenotypes.
A drug that exhibits the ability to overcome multiple drug resistance could be employed as a chemotherapeutic agent either alone or in combination with other drugs. The potential advantages of using such a drug in combination with chemotherapy would be the need to employ fewer toxic compounds in the combination, cost savings, and a synergistic effect leading to a treatment regime involving fewer treatments.
The commonly used chemotherapeutic agents are classified by their mode of action, origin, or structure, although some drugs do not fit clearly into any single group. The categories include alkylating agents, anti-metabolites, antibiotics, alkaloids, and miscellaneous agents (including hormones). Agents in the different categories have different sites of action.
Antibiotics are biologic products of bacteria or fungi. They do not share a single mechanism of action. The anthracyclines daunorubicin and doxorubicin (DOX) are some of the more commonly used chemotherapeutic antibiotics. The anthracyclines achieve their cytotoxic effect by several mechanisms, including inhibition of topoisomerase II; intercalation between DNA strands, thereby interfering with DNA and RNA synthesis; production of free radicals that react with and damage intracellular proteins and nucleic acids; chelation of divalent cations; and reaction with cell membranes. The wide range of potential sites of action may account for the broad efficacy as well as the toxicity of the anthracyclines (Young et al., 1985).
The anthracycline antibiotics are produced by the fungus Streptomyces peuceitius var. caesius. Although they differ only slightly in chemical structure, daunorubicin has been used primarily in the acute leukemias, whereas doxorubicin displays broader activity against human neoplasms, including a variety of solid tumors. The clinical value of both agents is limited by an unusual cardiomyopathy, the occurrence of which is related to the total dose of the drug; it is often irreversible. In a search for agents with high anti-tumor activity but reduced cardiac toxicity, anthracycline derivatives and related compounds have been prepared. Several of these have shown promise in the early stages of clinical study, and some, like epirubicin and idarubicin, are used as drugs. Epirubicin outsells doxorubicin in Europe and Japan, but it is not sold in the U.S.
The anthracycline antibiotics have tetracycline ring structures with an unusual sugar, daunosamine, attached by glycosidic linkage. Cytotoxic agents of this class all have quinone and hydroquinone moieties on adjacent rings that permit them to function as electron-accepting and donating agents. Although there are marked differences in the clinical use of daunorubicin and doxorubicin, their chemical structures differ only by a single hydroxyl group on C14. The chemical structures of daunorubicin and doxorubicin are shown in FIG. 1.
Doxorubicin""s broad spectrum of activity against most hematological malignancies as well as carcinomas of the lung, breast, and ovary has made it a leading agent in the treatment of neoplastic disease (Arcamone, 1981; Lown, 1988; Priebe, 1995). Since the discovery of daunorubicin and doxorubicin (FIG. 1), the mechanistic details of the anti-tumor activity of anthracycline antibiotics have been actively investigated (Priebe, 1995a; Priebe, 1995b; Booser, 1994).
Unfortunately, concomitant with its anti-tumor activity, DOX can produce adverse systemic effects, including acute myelosuppression, cumulative cardiotoxicity, and gastrointestinal toxicity (Young et al., 1985). At the cellular level, in both cultured mammalian cells and primary tumor cells, DOX can select for multiple mechanisms of drug resistance that decrease its chemotherapeutic efficacy. These mechanisms include P-gp-mediated MDR and MPR-rediated MDR, characterized by the energy-dependent transport of drugs from the cell (Bradley et al., 1988), and resistance conferred by decreased topoisomerase II activity, resulting in the decreased anthracycline-induced DNA strand scission (Danks et al., 1987; Pommier el al., 1986; Moscow et al., 1988).
Among the potential avenues of circumvention of systemic toxicity and cellular drug resistance of the natural anthracyclines is the development of semi-synthetic anthracycline analogues which demonstrate greater tumor-specific toxicity and less susceptibility to various forms of resistance.
The present invention seeks to overcome drawbacks inherent in the prior art by providing compositions of agents that display increased cytotoxicity when compared with doxorubicin and can prevent and/or overcome multi-drug resistance and exhibit reduced cardiotoxicity. This invention involves novel compounds that have utility as anti-tumor and/or chemotherapeutic drugs, methods of synthesizing these compounds and methods of using these compounds to treat patients with cancer. The invention is based on the discovery that anthracycline derivatives with substitutions attached to their C-3xe2x80x2 or C-4xe2x80x2 carbons in the sugar moiety have a surprisingly strong ability to kill multi-drug resistant tumor cells.
New anthracycline-based agents designed to interact and crosslink with DNA have been synthesized. These analogs contain substitutions at the C-3xe2x80x2 or C-4xe2x80x2 sugar moiety. Synthesized compounds displayed activity significantly higher than that of parental daunorubicin or doxorubicin. In brief, in vitro the compounds WP755, WP756, WP757, WP758, WP775, WP778, WP784, WP786, WP790, WP791 modified at the C-3xe2x80x2 and WP744, WP783 and WP750 modified at the C-4xe2x80x2 were significantly more effective as measured by resistance index (RI) (Table 2). The RI values for the 3xe2x80x2-O-substituted analogs vary from 1.2 to 36 and are low when compared to the RI value of 253 and  greater than 200 for DOX, wherein, a higher RI value indicates that a compound is less effective against MDR. Similarly, RI values were very low (1.4-D8.8) for 4xe2x80x2-O-alkylated analogs whereas RI values for DOX varied from  greater than 42.6 to  greater than 200 for MDR1 type of resistance and from 10 to 16 for the MRP form of resistance. Lower RI values indicate greater efficacy of the drug against MDR tumors. The inventors also designed and synthesized other analogs. The observed activity and high potency against multi-drug resistant tumors indicate that these analogs are different from the parental drugs like doxorubicin and daunorubicin.
The inventors synthesized a series of analogs substituted at the aromatic ring of the C-3xe2x80x2-substituent which were then combined with modifications at the aglycon moiety. The inventors discovered that substitution at the aromatic ring increased the potency and alter the mechanism of action of the drugs making them significantly more active than doxorubicin. The mechanism of action of this class of drugs might involve direct interaction of the aromatic ring with cellular targets like DNA, topoisomerase II and topoisomerase I. Substitution of a benzyl ring at C-3xe2x80x2 modifies drug interaction with P-glycoprotein, and subsequently the resistance index, thereby makes these compounds more effective against MDR tumors in comparison to the parent drug. In vitro evaluation identified the following C-3xe2x80x2 substituted anthracycline analogs: WP831, WP791, WP790, WP786, WP785, WP784, WP780, WP778, WP775, WP774, WP765, WP758, WP757, WP756 and WP755 as unusually effective cytotoxic agents when compared to DOX.
The inventors also synthesized anthracycline analogs with substitutions at the C-4xe2x80x2 sugar and demonstrated that these analogs overcome both, (a) multi-drug resistance (MDR) caused by overexpression of the MDR1 gene and (b) MDR-associated protein (MRP)-related resistance caused by overexpression of the MRP gene. Such modifications also contribute to the drugs ability to circumvent others forms of drug resistance and increased bioavailability. An increased steric hindrance at C-4xe2x80x2 in doxorubicin might reduce drug interaction with P-glycoprotein and MRP and in combination, the increased lipophilicity caused by introduction of aromatic ring further contributes to increase intracellular drug concentration in MDR cells. Such modifications alter cellular uptake and retention of the drugs without affecting interaction with cellular targets, which results in cytotoxic effects. In vitro evaluation identified the following C-4xe2x80x2 substituted anthracycline analogs: WP799, WP797, WP794, WP787, WP783, WP750, WP744, WP727, WP764 and WP571 as unusually potent cytotoxic agents when compared to DOX.
The anthracycline compounds bearing the C-3xe2x80x2 substitutions have among others, O-Benzyl, N-Benzyl or S-Benzyl substitutions where the phenyl, an aromatic ring of the benzyl group is substituted. The compounds bearing C-4xe2x80x2 substitutions at the sugar have among others, O-Benzyl, N-Benzyl or S-Benzyl substitutions where the benzyl group is a substituted or an unsubstituted benzene group. These compounds are depicted in FIGS. 2-25. These compounds exhibit cytotoxic activity substantially different from the activities of doxorubicin or daunorubicin. These compounds are active against doxorubicin resistant tumors and/or are usually similar or more cytotoxic than doxorubicin against sensitive tumors.
In some specific embodiments, the C-3xe2x80x2-substituted anthracycline compounds of the present invention have the general formula: 
wherein, R1 denotes any suitable group or combination of groups that form but are not limited to a nucleic acid intercalator or binding compound; a topoisomerase inhibitor, including but not limited to, an alkyl chain; a (xe2x80x94COCH2R13) group; or a (xe2x80x94C(OH)xe2x80x94CH2R13); wherein, R13 is a hydrogen (xe2x80x94H) group or a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); an alkoxy group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an aryl group having 1-20 carbon atoms; a fatty acyl group having the general structure (xe2x80x94Oxe2x80x94CO(CH2)nCH3) wherein n=an integer from 1 to about 20; or a fatty acyl group having the general structure (xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3) wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; or a chain(R) such as xe2x80x94OCOxe2x80x94(CH2)nxe2x80x94CH2NH2; or OCOxe2x80x94(CH2)nxe2x80x94CO2H and its salts; each of R2 and R3 is, independently of the other, a hydrogen (xe2x80x94H), a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); R4 is a hydrogen (xe2x80x94H) group; a methoxy group (xe2x80x94OCH3); a hydroxyl group (xe2x80x94OH); or a halide; each of Y1 and Y2 is, independently of the other, a double bonded oxygen, sulphur, or nitrogen atom; Z is a xe2x80x94H; xe2x80x94OH; a xe2x80x94CO2H group; or a xe2x80x94CO2R group; R7, R8, are, independently, xe2x80x94H; xe2x80x94OH; a halide; xe2x80x94OR19; xe2x80x94SH; xe2x80x94SR19; xe2x80x94NH2; xe2x80x94NHR19; xe2x80x94N(R19)2; xe2x80x94CH3; and R7 can additionally be a saccharide; wherein R19 is an alkyl chain; an alkylating moiety; a cycloalkyl chain; a cyclic ring; or a hydrogen; R9 can be xe2x80x94H; xe2x80x94CH3; alkyl; aryl; CH2OH, CH2F; R10, R11 and R12 are, independently, xe2x80x94H; xe2x80x94OH; a halide; xe2x80x94OR; xe2x80x94SH; xe2x80x94SR; xe2x80x94NH2; xe2x80x94NHR; xe2x80x94N(R)2; xe2x80x94CH3; one of R5 and R6 is a xe2x80x94H; one of R5 and R6 is a X-alkyl-aromatic-ring (AAR) substituent such as xe2x80x94XAAR, wherein, A is an alkyl group and wherein, AR is an substituted phenyl ring; or a substituted five-member ring; or a heteroatomic five-member ring; or a heteroatomic six-member ring such as a pyridine ring; of the form; 
wherein, R14-R18 are independently a (xe2x80x94H) group; a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); a nitro group (xe2x80x94NO2), an amine group (xe2x80x94NH2), a halide; an alkoxy group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an aryl group having 1-20 carbon atoms; an alkyl-amino group; an alkyl-thio group; a cyano group (CN, SCN); an xe2x80x94CO2H group; an xe2x80x94CO2R group; and the aromatic ring may be disubstituted, trisubstituted, tetrasubstituted or pentasubstituted; and X is a xe2x80x94O, xe2x80x94N or xe2x80x94S, or xe2x80x94SO, or xe2x80x94SO2 group; and A is (CH2)n where n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein, if R5 is a XAAR substituent R6 is not and if R6 is a XAAR substituent R5 is not.
In other specific embodiments, the C-4xe2x80x2 substituted anthracycline compounds of the present invention have the general formula: 
wherein, R1 denotes any suitable group or combination of groups that form but are not limited to a nucleic acid intercalator or binding compound; a topoisomerase inhibitor, including but not limited to, an alkyl chain; a (xe2x80x94COCH2R13) group; or a (C(OH)xe2x80x94CH2R13) wherein, R13 is a hydrogen (xe2x80x94H) group or a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); an alkoxy group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an aryl group having 1-20 carbon atoms; a fatty acyl group having the general structure(xe2x80x94Oxe2x80x94CO(CH2)nCH3) wherein n=an integer from 1 to about 20; or a fatty acyl group having the general structure (xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3), wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; or a chain(R) such as xe2x80x94OCOxe2x80x94(CH2)nxe2x80x94CH2NH2 ; or OCOxe2x80x94(CH2)nxe2x80x94CO2H and its salts; each of R2 and R3 is, independently of the other, a hydrogen (xe2x80x94H), a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); R4 is a hydrogen (xe2x80x94H) group; a methoxy group (xe2x80x94OCH3); a hydroxyl group (xe2x80x94OH); or a halide; each of Y1 and Y2 is, independently of the other, a double bonded oxygen, sulphur, or nitrogen atom; Z is a xe2x80x94H; xe2x80x94OH; a xe2x80x94CO2H group; or a xe2x80x94CO2R group; R5, R6, are, independently, xe2x80x94H; xe2x80x94OH; a halide; xe2x80x94OR19; xe2x80x94SH; xe2x80x94SR19; xe2x80x94NH2; xe2x80x94NHR19; xe2x80x94N(R19)2; xe2x80x94CH3; wherein R19 is an alkyl chain; an alkylating moiety; a cycloalkyl chain; a cyclic ring; or a hydrogen; and R6 can additionally be a an alkylating moiety; R9 can be xe2x80x94H; xe2x80x94CH3; alkyl; aryl; CH2OH, CH2F; R10, R11 and R12 are, independently, xe2x80x94H; xe2x80x94OH; a halide; xe2x80x94OR; xe2x80x94SH; xe2x80x94SR; xe2x80x94NH2; xe2x80x94NHR; xe2x80x94N(R)2; xe2x80x94CH3; one of R7 and R8 is a xe2x80x94H; one of R7 and R8 is a X-alkyl aromatic-ring (AAR) substituent such as xe2x80x94XAAR, wherein, A is an alkyl group and wherein, AR is an unsubstituted phenyl ring; or a substituted phenyl ring; or a substituted five-member ring such as a pyridine ring; or a heteroatomic five-member ring, of the general form; 
wherein, R14-R18 are independently a (xe2x80x94H) group; a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); a nitro group (xe2x80x94NO2), an amine group (xe2x80x94NH2), a halide; an alkoxy group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an aryl group having 1-20 carbon atoms; an alkyl-amino group; an alkyl-thio group; a cyano group (CN, SCN); an xe2x80x94CO2H group; an xe2x80x94CO2R group; and the aromatic ring may be disubstituted, trisubstituted, tetrasubstituted or pentasubstituted; and X is a xe2x80x94O, xe2x80x94N or xe2x80x94S, or xe2x80x94SO, or xe2x80x94SO2 group; and A is (CH2)n where n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein if R7 is a XAAR substituent R8 is not and if R8 is a XAAR substituent R7 is not.
Certain specific embodiments of the anthracyclines of the invention are shown in FIGS. 2-25.
The present application also comprises methods of preparing novel substituted sugar substrates and their use in the synthesis of the novel anthracycline analogs described in this invention. In certain embodiments the method for the synthesis of 4xe2x80x2-O-benzylated sugars is described. These 4-O-benzylated sugars may comprise one of two main classes; glycal sugars or 1-O-silyalated sugars. Examples of the benzylated sugars encompassed by this invention are WP567, WP735, WP736, WP819, W0820, WP821, WP822, WP823, WP824 and WP825.
Related embodiments describe the method for synthesizing glycals using various bases including but not limited to NaH. Other related embodiments describe the synthesis of glycals using various solvents including but not limited to DMF. Yet other related embodiments describe the synthesis of glycals using various alkylating agents including but not limited to benzyl chloride and benzyl bromide.
In certain embodiments, the method for the synthesis of amine containing analogs of anthracyclines is described. Further embodiments describe the use of substituted sugar azides for the synthesis of said amine containing anthracyclines wherein the azido substitution can be at the 1xe2x80x2, 2xe2x80x2, 3xe2x80x2, 4xe2x80x2 or 5xe2x80x2 position on the sugar. The azide group serves as a masked or neutral form of amine substituent allowing for a coupling reaction (explained in Example 2, Procedure A). This allows the generation fo selective conditions to reduce azides (explained in Example 2, Procedure B). In one example the amine containing anthracyclines synthesized by this procedure are 14-OH analogs similar to doxorubicin, epirubicin or daunoribicin such as WP744 and WP769. The azido sugar used in the preparation of this compound is WP819 (explained in Example 1, Procedure A and B).
This procedure also allows the use of 14-O-blocked aglycons as these blocked groups survive the steps in which azides are reduced and can be selectively removed at a later satge (Example 2, Procedure B).
The present application comprises methods of preparing substituted anthracyclines and the preparation of important sugar substrates. In devising the synthetic schemes and compounds of the present invention, the inventors have created a variety of novel compounds. These compounds and their methods of synthesis are described elsewhere in the specification, examples and figures and are given xe2x80x9cWPxe2x80x9d numbers. The structure of a compound designated with a xe2x80x9cWPxe2x80x9d number is ascertainable by reviewing the specification and figures. Exemplary specific anthracycline compounds that are encompassed by the invention are WP831, WP791, WP790, WP787, WP786, WP785, WP784, WP780, WP778, WP775, WP774, WP765, WP764, WP758, WP757, WP756, WP755, WP799, WP797, WP794, WP783, WP750, WP744, WP727 and WP571. Exemplary specific sugar substrates that are encompassed by the invention are WP567, WP735, WP736, WP819, WP820, WP821, WP822, WP823, WP824, WP825.
The invention also considers methods of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of the contemplated substituted anthracycline compounds and therapeutic kits comprising, in suitable container means, a pharmaceutically acceptable composition comprising the contemplated substituted anthracycline compounds.